The botulinum neurotoxins (BoNT) are the most toxic proteins known for humans and the causative agent of botulism. BoNTs are organized into three domains that are involved in catalysis (LC), translocation (HCT), and receptor binding (HCR). There are now eight BoNT serotypes (A-H) based upon limited cross serotype protection of ?-sera against each BoNT serotype. Currently, there is no licensed vaccine against botulism and the experimental penta-serotype toxoid vaccine previously available from the CDC for at-risk populations was discontinued in 2011. Thus, there is a need to develop a potent and effective BoNT vaccine against all BoNT serotypes to protect at-risk humans from exposure, including civilians in harm's way, first responders, the military, and researchers. Several approaches to generate new BoNT vaccines have not yielded a vaccine product. Two published vaccine approaches against botulism developed through collaborations between the Barbieri and Johnson laboratories include an HCR subunit vaccine that protected against challenge by the seven BoNT serotypes, and a full-length, atoxic BoNT (M-BoNT) vaccine that protected against BoNT/A challenge. HCRs are stable products made in large quantity, are nontoxic and not Select Agent regulated, thus making product development straightforward. The goal of this study is to optimize the HCR and M-BoNT vaccine platforms towards the generation of a pan-BoNT protective vaccine against foodborne and inhalation botulism. The hypothesis is that native BoNT or HCR derivatives that have been mutated to eliminate cell binding and catalytic activity will improve vaccine potency against all BoNT serotypes compared to toxoid vaccines, while enhancing safety and reducing vaccine side effects. The specific aims will utilize the complementary research expertise of both the Barbieri and Johnson laboratories. The aims will engineer a pan-BoNT serotype protective vaccine against botulism, using the mouse challenge model. Optimized vaccines will be tested for potency in food borne- and inhalation- models of botulism. These studies will produce the next generation vaccine against botulism and a strategy for rapid response to the release of BoNT variants.